Commonly abused drugs are diverse in their chemical and pharmacological profiles, but they act on neural systems which support attention and consciousness, and their repetitive use leads to a state of dependence, characterized by withdrawal syndromes. While the neurochemical events leading to dependence remain only partially understood, there is increasing evidence for the involvement of epigenetic mechanisms such as DNA methylation. Studies our lab have identified a critical role for the cysteine/glutamate uptake transporter EAAT3 (excitatory amino acid transporter 3) in controlling redox and methylation status in human neuronal cells. EAAT3-mediated cysteine transport is stimulated by dopamine, involving the D4 dopamine receptor, which has been linked to the frequency of drug abuse. Preliminary studies show that EAAT3- mediated cysteine uptake is also sensitive to opiates and ethanol, as well as nicotine and cannabinoids, suggesting that it may serve as a nexus for the epigenetic effects of abused drugs. To investigate this possibility, we will carry out a detailed investigation of the influence of selected drugs of abuse on redox and methylation status of cultured human neuroblastoma cells and rat-derived primary cultured neurons. We will measure the dose and time-dependent effects of morphine, amphetamine and alcohol on the cellular uptake of [35S]-cysteine, which primarily reflects EAAT3 activity. Under the same conditions, we will measure drug-induced changes of intracellular thiols (reduced and oxidized forms of glutathione (GSH and GSSG), cysteine and homocysteine) as well as methylation-related metabolites methionine, S- adenosylmethionine (SAM), S-adenosylhomocysteine (SAH) and cystathione. The ratio of GSH to GSSG is an indication of cellular redox status, while SAM to SAH is a reflection of methylation capacity. Changes in global DNA methylation and CpG island methylation status will be measured to assess the epigenetic effects of each drug.. Based upon preliminary data, we hypothesize that these parameters will be responsive to the selected drugs of abuse, and that a new steady-state redox and methylation status will be attained in their sustained presence, representing adaptive neuronal responses to their presence. We will monitor time-dependent changes in cysteine uptake and cellular redox/methylation status upon drug removal, representing a reversal of the initial adaptive responses, analogous to withdrawal and evaluate the influence of a previous exposure on subsequent redox and methylation responses to the same or different drug (i.e. sensitization or tolerance and cross-reactivity). Finally, we will use qRT-PCR to evaluate changes in transcription of selected redox and methylation-related genes, which may mediate adaptive responses to drugs of abuse during their presence and/or contribute to the withdrawal state in their absence. Experimental results for each drug will be incorporated into a computational model of the relevant metabolic pathways. This exploratory project will therefore examine a novel hypothesis related to the mechanisms of drug addiction. PUBLIC HEALTH RELEVANCE: The proposed project will carry out a detailed study of how morphine, methamphetamine, alcohol each affect sulfur metabolism and antioxidant status in cultured human neuroblastoma cells and primary cultured neurons. The results will provide an entirely new way of thinking about how drugs of abuse affect the brain and could lead to improved ways to treatment to treat drug addiction.